Method and composition for diagnosis of melanocytic lesions

ABSTRACT

This invention relates to monoclonal antibodies that recognize an antigen specific to melanocytic lesions. These antibodies are useful in methods of isolating melanoma cells and diagnosing melanocytic lesions. These antibodies are also useful for immunotherapy against melanoma.

I. FIELD OF THE INVENTION

This invention relates to melanoma-associated antigens, antibodiesagainst melanoma-associated antigens, and diagnostic and therapeuticmethods concerning melanoma.

II. BACKGROUND OF THE INVENTION

Human malignant melanoma usually starts as harmless moles that undergoradial to invasive growth and end in the destructive stage of metastaticmelanoma. Melanoma usually resists chemotherapy and radiotherapy.Surgery is the most effective treatment. Early diagnosis is required forsurgery to be effective, which is unfortunately hampered by the lack ofmarkers that are sensitive and specific for melanoma.

The commonly used antibodies for immuno-histochemical diagnosis ofmelanoma are HMB-45, anti-S-100 and NKI/C3 (for a review, please seeSmoller B R, “Immunohistochemistry in the Diagnosis of MelanocyticNeoplasms”, in PATHOLOGY: State of the Art Reviews, 2:371-383, 1994).

HMB-45 has been shown to react with the melanoma-associated antigengp-100. Adema G J, de-Boer A J, Vogel A M, Loenen W A, Figdor C G:Molecular characterization of the melanocyte lineage-specific antigengp100. J. Biol. Chem. 1994; 269:20126-20133. HMB-45 reacts withmelanoma, junctional nevi, dysplastic nevi, spindle cell and epitheloidcell nevi, congenital nevi and blue nevi. Gown A M, Vogel A M, Hoak D,Gough F, McNutt M A: Monoclonal antibodies specific for melanocytictumors distinguish subpopulations of melanocytes. Am. J. Pathol. 1986;123:195-203; Esclamado R M, Gown A M, Vogel A M. Unique proteins definedby monoclonal antibodies specific for human melanoma. Some potentialclinical applications. Am. J. Surg. 1986; 152:376-385; Palazzo J, DurayP H: Typical, dysplastic, congenital, and Spitz nevi.: a comparativeimmuno-histochemical study. Hum Pathol 1989; 20:341-346; Smoller B R,McNutt N S, Hsu A: HMB-45 recognizes stimulated melanocytes. J. Cutan.Pathol. 1989; 16:49-53; Smoller B R, McNutt N S, Hsu A: HMB-45 stainingof dysplastic nevi. Support for a spectrum of progression towardmelanoma. Am J Surg Pathol 1989; 13:680-684; Sun J, Morton T H Jr., GownA M: Antibody HMB-45 identifies the cells of blue nevi. Animmuno-histochemical study on paraffin sections. Am J Surg Pathol 1990;14:748-751. HMB-45 is routinely used as a marker to differentiatemelanoma from nonmelanocytic cancer cells. Herrera G A, Hancock C.Specificity of antibody HMB-45. Arch. Pathol. La. Med. 1992;116:900-901. However, diagnosis with HMB-45 leaves a sizable numbers ofmelanomas undetected because the sensitivity of HMB-45 is between 67%and 93%. Wick M R, Swanson P E, Rocamora A. Recognition of malignantmelanoma by monoclonal antibody HMB-45. An immuno-histochemical study of200 paraffin-embedded cutaneous tumors. J Cutan Pathol 1988; 15:201-207;Ordonez N G, Ji X L, Hickey R C: Comparison of HMB-45 monoclonalantibody and S100 protein in the immuno-histochemical diagnosis ofmelanoma. Am. J. Clin. Pathol. 1988; 90:385-390.

Antibodies to S-100 protein have higher sensitivity than HMB-45 withmalignant melanoma varieties. Nakajima T, Watanabe S, Sato Y, Kameya T,Shimosato Y, Ishihara K: Immuno-histochemical demonstration of S100protein in malignant melanoma and pigmented nevus and its diagnosticapplication. Cancer 1982; 50:912-918; Kindblom L G, Lodding P, RosengrenL, Baudier J, Haglid K: S100 protein in melanocytic tumors. Animmuno-histochemical investigation of benign and malignant melanocytictumors and metastases of malignant melanoma and a characterization ofthe antigen in comparison to human brain. Acta. Pathol. Microbiol.Immunol. Scand. 1984; 92:219-230; Springall D R, Gu J, Cocchia D,Michetti F, Levene A, Levene M M et al: The value of S100 immunostainingas a diagnostic tool in human malignant melanomas. A comparative studyusing S100 and neuron-specific enolase antibodies. Virchows Arch.Pathol. Anat. Histopathol. 1983; 400:331-343; Cochran A J, Wen D R,Herschman H R, Gaynor R B: Detection of S100 protein as an aid to theidentification of melanocytic tumors. Int. J. Cancer 1982; 30:295-297;Gatter K C, Ralfkiaer E, Skinner J, Brown D, Heryet A, Pulford K A etal: An immunocytochemical study of malignant melanoma and itsdifferential diagnosis from other malignant tumors. J Clin Pathol 1985;38:1353-1357; Hachisuka H, Sakamoto F, Nomura H, Mori O, Sasai Y:Immuno-histochemical study of S100 protein and neuron specific enolase(NSE) in melanocytes and the related tumors. Acta Histochem. 1986;80:215-223; Argenyi Z B, Cain C, Bromley C, Nguyen A V, Abraham A A,Kerschmann R et al: S100 protein-negative malignant melanoma: fact orfiction? A light-microscopic and immuno-histochemical study. Am. J.Dermatopathol. 1994; 16:233-240 (11-17). However, anti-S100 antibodiesare notoriously unspecific. Anti-S100 antibodies have been shown tostain benign cells such as salivary and sweat glands, skeletal andcardiac muscle, histiocytes, Schwann, cells, lipocytes, chondrocytes,astrocytes, oligodendrocytes (Kindblom L G, Lodding P, Rosengren L,Baudier J, Haglid K: S100 protein in melanocytic tumors. Animmuno-histochemical investigation of benign and malignant melanocytictumors and metastases of malignant melanoma and a characterization ofthe antigen in comparison to human brain. Acta. Pathol. Microbiol.Immunol. Scand. 1984; 92:219-230; Stefansson K, Wollmann R L, Moore B W,Arnason B G. S100 protein in human chondrocytes. Nature 1982; 295:63-64;Stefansson K, Wollmann R L, Moore B W: Distribution of S100 proteinoutside the central nervous system. Brain Res. 1982; 234:309-317) andtumors arising from these cells (Tabuchi K, Moriya Y, Furuta T, OhnishiR, Nishimoto: A S100 protein in human glial tumors. Qualitative andquantitative studies. Acta Neurochir Wien 1982; 65:239-251; Vanstapel MJ, Gatter K C, de Wolf Peeters C, Mason D Y, Desmet V D. New sites ofhuman S100 immunoreactivity detected with monoclonal antibodies. Am JClin Pathol 1986; 85:160-168). Anti-S100 antibodies have also been shownto stain many non-melanoma malignant tumor tissues. Drier J K, Swanson PE, Cherwitz D L, Wick M R: S100 protein immunoreactivity in poorlydifferentiated carcinomas. Immuno-histochemical comparison withmalignant melanoma. Arch. Pathol. Lab. Med. 1987; 111:447-452.

NKI/C3 antibody also reacts with a broad range of benign and malignantneoplasms. Mackie R M, Campbell I, Turbitt M L. Use of NK1 C3 monoclonalantibody in the assessment of benign and malignant melanocytic lesions.J Clin Pathol 1984; 37:367-372; Vennegoor C, Calafat J, Hageman P, vanBuitenen F, Janssen H, Kolk A et al. Biochemical characterization andcellular localization of a formalin-resistant melanoma-associatedantigen reacting with monoclonal antibody NKI/C-3. Int. J. Cancer 1985;35:287-295. These unspecific activities limit the usefulness of NKI/C3antibody in the diagnosis of melanocytic lesions.

KBA.62 antibody is comparable to HMB-45 in its sensitivity to melanoma,but is less specific than HMB-45 in that KBA.62 stains squamous cellcarcinoma and basal cell carcinomas. Cohen Knafo E, al Saati T, Aziza J,Ralfkiaer E, Selves J, Gorguet B et al: Production and characterisationof an antimelanoma monoclonal antibody KBA.62 using a new melanoma cellline reactive on paraffin wax embedded sections. J Clin Pathol 1995;48:826-831.

III. SUMMARY OF THE INVENTION

Within the scope of this invention, Applicant prepared a melanomaspecific monoclonal antibody, SM5-1, using a subtractive immunizationprotocol. Immuno-histochemical staining of paraffin-embedded specimensof melanomas (primary melanomas and metastases) and skin nevi samplesshowed that SM5-1 reacts with all nevi and malignant melanocyticlesions. On the other hand, SM5-1 does not react with non-activatedepidermal melanocytes, non-melanocytic tumor cells listed in Table 2,keratinocytes, endothelial cells, smooth muscle cells and peripheralnerves. These results indicate that SM5-1 is extremely sensitive andhighly specific for melanocytic lesions.

A hybridoma secreting SM5-1 antibody was deposited with America TypeCulture Collection on Oct. 20, 1998 under ATCC Accession No. HB-12588.

A. Antigen

Thus, in one aspect, this invention features an isolated, purified,enriched, or synthetic antigen which binds specifically to SM5-1antibody. In addition, this antigen is characterized in that it ispresent on the membrane and in the cytoplasm of all human melanomacells, including human nevi, primary melanoma cells, and metastaticmelanoma cells. Furthermore, this antigen is characterized in that it isnot detectably expressed in normal non-activated human melanocytic cellsas tested by SM5-1 antibody. Neither is this antigen detectablyexpressed in human tumor cells listed in Table 2 as tested by SM5-1antibody. This antigen may be obtained from natural sources (e.g., cellsor extracellular fluid) or by chemical synthesis.

The antigen of this invention can be used to prepare a melanoma vaccineto elicit a specific immunological response against melanoma fortreating or preventing the development or spread of melanoma. For thispurpose, the melanoma antigen can be mixed with an adjuvant (an agentwhich increases the immune response for the immunization procedure),including, but not limited to, Freund's complete adjuvant (emulsion ofmineral oil, water, and mycobacterial extracts), Freund's incompleteadjuvant (emulsion of water and oil only), mineral gels, e.g. aluminumhydroxide gels, surface active substances such as lysolecithin,polyanions, peptides, BCG (Bacillus Calmette-Guerin). The routes ofimmunization include, among others, intradermal, subcutaneous,intramuscular, intraperitoneal, intraspleen, intralymphnode, intratumor,intravascular and intracranial injections. A series of injections can begiven to achieve high antibody titers and/or T cell response. Forexample, the immunization is performed by injecting the antigen two,three, four or more times parenterally, e.g. intraperitoneally and/orsubcutaneously, in regular or irregular intervals of a few days, e.g.three to seven days, up to several months, for example four weeks.

By “isolated” in reference to a polypeptide (e.g., an antigen or anantibody) is meant a polypeptide that is isolated from a natural source(e.g., cells, including tumor cells, or extracellular fluid) or that issynthesized. The isolated polypeptides of the present invention areunique in the sense that they are not found in a pure or separated statein nature. Use of the term “isolated” indicates that a naturallyoccurring sequence has been removed from its normal cellularenvironment. Thus, the polypeptide may be in a cell-free solution orplaced in a different cellular environment. The term does not imply thatthe polypeptide is the only amino acid chain present, but that it is thepredominate amino acid chain present (at least 10-20% more than anyother sequence) and is essentially free (about 90-95% pure at least) ofnon-amino acid material naturally associated with it.

By “enriched” in reference to a polypeptide (e.g., an antigen or anantibody) is meant that the specific polypeptide constitutes asignificantly higher fraction (2-5 fold) of the total of amino acidchains present in the cells or solution of interest than in normal ordiseased cells or in the cells from which the sequence was taken. Thiscould be caused by a person by preferential reduction in the amount ofother amino acid chains present, or by a preferential increase in theamount of the specific polypeptide of interest, or by a combination ofthe two. However, it should be noted that enriched does not imply thatthere are no other amino acid sequences present, just that the relativeamount of the polypeptide of interest has been significantly increased.The term “significantly” here is used to indicate that the level ofincrease is useful to the person making such an increase, and generallymeans an increase relative to other amino acid chains of about at least2 fold, more preferably at least 5 to 10 fold or even more. The termalso does not imply that there is no amino acid chains from othersources.

By “purified” in reference to a polypeptide (e.g., an antigen or anantibody) does not require absolute purity (such as a homogeneouspreparation); instead, it represents an indication that the polypeptideis relatively purer than in the natural environment (compared to thenatural level this level should be at least 2-5 fold greater, e.g., interms of mg/ml). Purification of at least one order of magnitude,preferably two or three orders, and more preferably four or five ordersof magnitude is expressly contemplated. The substance is preferably freeof contamination at a functionally significant level, for example 90%,95%, or 99% pure.

B. Antibody and Other Affinity Molecules

In a second aspect, the present invention features an affinity moleculethat recognizes the antigen recognized by the SM5-1 antibody. Affinitymolecules include, but are not limited to, antibody, peptide, nucleicacid aptamer, and other organic polymers with specific binding affinityto the antigen. In a preferred embodiment, the affinity molecule is amonoclonal antibody. Different monoclonal antibodies of this inventionmay recognize different epitopes on the antigen. Preferably, themonoclonal antibody of this invention is purified, isolated or enriched.

In a preferred embodiment, this monoclonal antibody (i) recognizes anantigen on the membrane and in the cytoplasm of human nevi, primarymelanoma cells, and metastatic melanoma cells, (ii) does not recognizean antigen on normal non-activated human melanocytes, and (iii) does notrecognize an antigen on human tumor cells listed in Table 2.

In another preferred embodiment, the present invention contemplatesmonoclonal antibodies that correspond to the monoclonal antibodyproduced by ATCC HB-12588, and, in a particularly preferred embodiment,the ATCC HB-12588 antibody. One antibody corresponds to another antibodyif they both recognize the same or overlapping antigen binding sites asdemonstrated by, for example, a binding inhibition assay.

The antibodies obtained and employed may be any one of theimmunoglobulin classes, such as IgA, IgD, IgE, IgG and IgM, and any oneof the subclasses of the various classes. In addition, whole antibodiesor fragments of the antibodies may be employed, such as FAB, F(ab)′₂,Fv, or the like. The subject immortalized B-lymphocytes or other cells,for example T-cells, which provide monoclonal antibodies specific forthe subject epitope, can be used as a source of DNA, either genomic orcDNA, for expression of the ligand heavy chains of the monoclonalantibodies in procaryotes or eucaryotes. The resulting products may thenbe used as receptors for binding to the subject epitopes.

The present invention also provides derivatives of antibodies thatpossess the same binding specificity as the antibodies of the inventionas described above. These derivatives contain modifications in a regionthat is not important for antigen binding. These antibody derivativescan be obtained from antibodies according to the present invention byexchanging one or more constant domains and/or linkages with anothermolecule. Thus, for example, an exchange of constant domains can becarried out where, for example, an antibody of class IgM can beconverted into an antibody of class IgG with maintenance of its antigenspecificity. This and other isotype switches can be carried out bycell-biological or molecular-biological methods known to those skilledin the art, including, but are not limited to, that described in Rothmanet al., (1990) Mol. Cell. Biol., 10:1672-1679. In another example, theconstant domains of a murine antibody of this invention can be exchangedfor the constant domains of a human antibody. Methods of preparinghumanized antibody are applicable to the present invention, including,but not limited to, those methods described and cited in U.S. Pat. Nos.5,530,101 and 5,693,762, both entitled “Humanized immunoglobulins”, andWO 98/24884 entitled “Transgenic non-human animals capable of producingheterologous antibodies”, both of which are incorporated by referenceherein.

The monoclonal antibodies of the present invention can also be linkedwith another molecule, especially a label or a toxin, including, but notlimited to, enzymes such as peroxidase, toxins such as ricin or choleratoxin, and fluorescent and radioactive labels.

Monoclonal antibodies can be produced readily by one skilled in the artusing cell fusion techniques. Immortal, antibody-secreting cell linescan also be produced by techniques other than fusion, such as directtransformation of B-lymphocytes with oncogenic DNA, virus or othermeans. Different antigen sources can be used to challenge the normalB-lymphocyte population that is later converted to an immortal cellline. For example, the antigen immunoprecipitated by SM5-1 antibody canbe used as an immunogen to challenge the mammal (e.g., mouse, rat,hamster, etc.) used as a source for normal B-lymphocytes. Theantigen-stimulated B-lymphocytes are then harvested and fused to animmortal cell line or transformed into an immortal cell line by anyappropriate technique. Antibody-producing immortal cells can be screenedfor anti-melanoma cell antibody production by selecting clones that arestrongly reactive with the antigen or by selecting clones that competewith SM5-1 for reaction with melanoma cells and/or the isolated orpurified antigen.

Other than monoclonal antibodies, this invention also features peptides,nucleic acid aptamers and other molecules that recognize the antigenrecognized by the SM5-1 antibody. Methods of selecting affinity peptidesand aptamers are applicable to the present invention, including, but notlimited to, those methods described and cited in U.S. Pat. Nos.5,223,409, 5,475,096, 5,595,877, and 5,660,985, all of which areincorporated by reference herein.

C. Hybridoma Cell Lines

In a third aspect, the present invention features a continuous cell linethat produces an above-described antibody of the present invention. In apreferred embodiment, this cell line is a hybridoma. In anotherpreferred embodiment, this cell line is an immortal cell line.

D. Isolating Melanoma Cells and Other Cells Expressing the AntigenRecognized by SM5-1

The monoclonal antibodies and other affinity molecules of the presentinvention facilitate the isolation of melanoma antigen. One can usetechniques described in Chapter 13 of Harlow and Lane, “ImmunoaffinityPurification”, pages 511-552, ANTIBODIES—A LABORATORY MANUAL, ColdSpring Harbor Laboratory, 1988, incorporated by reference herein.

The monoclonal antibodies and other affinity molecules of the presentinvention facilitate the isolation of melanoma cells. Thus, in a fourthaspect, the present invention provides a method of producing apopulation of human cells enriched with human melanoma cells comprising:(a) providing a cell suspension from human tissue suspected ofcontaining melanoma cells; (b) contacting said cell suspension with amonoclonal antibody (or another affinity molecule) that recognizes theantigen recognized by the SM5-1 antibody; and (c) separating andrecovering from said cell suspension the cells bound by said antibody.

Human melanoma cells and other cells that express the antigen recognizedby SM5-1 can be separated from other cells with the antibodies of thisinvention using the indirect immune adherence (“panning”) techniques andthe fluorescence-activated cell sorting (FACS) techniques and othertechniques known to those skilled in the art. For example, the antibodybound to the cell can be labeled and then the cells separated by amechanical cell sorter that detects the presence of the label.Fluorescence-activated cell sorters are well known in the art.

In another example, the anti-melanoma antibody or another affinitymolecule is attached to a solid support. Various solid supports areknown to those skilled in the art, including, but not limited to,agarose beads, magnetic beads, polystyrene beads, hollow fiber membranesand plastic petri dishes. Cells that are bound by the antibody can beremoved from the cell suspension by simply physically separating thesolid support from the cell suspension.

Thus, in a fifth aspect, the present invention features isolated,purified or enriched human melanoma cells, e.g., a suspension of humancells comprising human melanoma cells substantially free of normal humanmelanoma cells and non-melanocytic tumor cells. The isolated melanomacells or other cells expressing the antigen recognized by SM5-1 can beemployed to produce anti-tumor antibodies, cytotoxic T cells andcellular vaccines against melanoma using techniques known to thoseskilled in the art, including, but not limited to, those described andcited in PCT publications WO 97/47271, WO 98/16238 and WO 98/04282, allof which are incorporated by reference herein.

E. Immuno-Histochemistry Diagnosis

The monoclonal antibodies and other affinity molecules of this inventioncan be used to screen patient samples for melanoma, including tissuebiopsy specimens, blood and other body fluid samples.

Biopsy specimens can be used for diagnosis of melanoma withimmunohistochemical staining techniques. The tissue sample may be fixedin formalin or other standard histological preservatives, dehydrated andembedded in paraffin as is routine in hospital pathology laboratory.Sections can be cut from the paraffin embedded material and mounted onglass slides or the sections can be prepared from cryo-preserved tissue.Alternatively, cytological preparations can be used. For example, cellsfrom the tissue sample can be fixed on a slide, typically by exposure toformalin in a buffer at physiologic pH, followed by suspension inacetone and pelleting onto gelatin-coated slides by centrifugation. Thecellular antigen can be localized, either by exposure to labeledantibody or by exposure to unlabeled antibody and a labeled secondaryantibody. The amount of the cell surface protein or antigen in thesample is proportional to the amount of bound label. In a preferredembodiment, SM5-1 is used in the immuno-histochemical evaluation ofmelanoma.

F. Serum Diagnosis

Aside from biopsy specimens, the melanoma antigen defined by themonoclonal antibodies of this invention can be detected in tissuesamples and bodily fluids by immunological methods known to thoseskilled in the art. Sample preparation will vary depending on the sourceof the biological sample. Tissue samples may be prepared by lysing thecells. Serum samples can be prepared by clotting whole blood andisolating the supernatant in accordance with well-known methods. Urine,bile fluid and other body fluid samples may be concentrated beforetesting. A wide variety of techniques and protocols exist for detectingan antigen in a sample suspected of containing the antigen. One can usetechniques described in Chapters 12 and 14 of Harlow and Lane,“Immunoblotting” and “Immunoassays”, pages 471-510 and 552-612,respectively, ANTIBODIES—A LABORATORY MANUAL, Cold Spring HarborLaboratory, 1988, incorporated by reference herein.

For example, two or more monoclonal antibodies can be used and theincubation with the individual antibodies can take place in varioussequences in homogeneous or heterogeneous phase. A signal is generateddue to binding of at least two monoclonal antibodies with the antigen tobe detected in the body fluid sample. The determination can take placeeither in homogeneous phase (e.g., agglutination test). Thedetermination can also take place in heterogeneous phase (e.g., sandwichimmunoassay). In both cases, at least two monoclonal antibodies are usedwhich bind to non-overlapping epitopes on the antigen.

A wide variety of labels can be used, such as radio-nuclids, enzymes,fluorescers, fluorescer-quencher combinations, chemiluminescers,magnetic particles, radiopaque dyes, and the like. These labels can bedirectly conjugated to the monoclonal antibody through a variety ofcovalently bonded linking groups and functionalities. Some of thetechniques involve having one of the members of the antigen-antibodycomplex bound to a support, such as a particle or vessel wall. Otherassays are performed in solution without a separation step, whereuncomplexed label is separated from complexed label. In a number ofassays, the antibody need not be labeled, such as in hemagglutination orwhere anti-immunoglobulin is employed and the anti-immunoglobulin islabeled, so as to provide for indirect labeling of the subjectmonoclonal antibody. Applicable assays include ELISA, RIA, EIA (Frye, etal., (1987) Oncogene 4:1153-1157) and the like.

The invention also concerns test kits for the qualitative andquantitative determination of the melanoma-associated antigen comprisingmonoclonal antibodies or other affinity molecules of the inventionand/or derivatives thereof and, optionally, other polyclonal ormonoclonal antibodies and/or adjuncts.

Test kits according to the invention for a radioimmunoassay contain, forexample, a suitable carrier, optionally freeze-dried solutions of one ormore polyclonal and/or monoclonal antibodies, solutions of aradioactively labeled antibody, standard solutions of the melanomaantigen, buffer solutions, and, optionally, polypeptides or detergentsfor preventing non-specific adsorption and aggregate formation,pipettes, reaction vessels, calibration curves, instruction manuals andthe like. One of the antibodies of the test kit is a monoclonal antibodyagainst the melanoma antigen.

Test kits according to the invention for an enzyme immunoassay contain,for example, a suitable carrier, optionally freeze-dried solutions ofone or more polyclonal and/or monoclonal antibodies, optionallyfreeze-dried or concentrated solutions of an enzyme- orbiotin-conjugated antibody, solutions of an enzyme-avidin conjugate ifbiotin-labeled antibody is used, enzyme substrate in solid or dissolvedform, standard solutions of the melanoma antigen, buffer solutions, and,optionally, polypeptides or detergents for preventing non-specificadsorption and aggregate formation, pipettes, reaction vessels,calibration curves, instruction manuals and the like. One of theantibodies of the test kit is a monoclonal antibody against the melanomaantigen.

The invention also concerns test kits for the qualitative andquantitative determination of the melanoma-associated antigen comprisingmonoclonal antibodies of the invention and/or derivatives thereof and,optionally, other polyclonal or monoclonal antibodies and/or adjuncts.

Test kits according to the invention for a radioimmunoassay contain, forexample, a suitable carrier, optionally freeze-dried solutions of one ormore polyclonal and/or monoclonal antibodies, solutions of aradioactively labeled antibody, standard solutions of the melanomaantigen, buffer solutions, and, optionally, polypeptides or detergentsfor preventing non-specific adsorption and aggregate formation,pipettes, reaction vessels, calibration curves, instruction manuals andthe like. One of the antibodies of the test kit is a monoclonal antibodyagainst the melanoma antigen.

Test kits according to the invention for an enzyme immunoassay contain,for example, a suitable carrier, optionally freeze-dried solutions ofone or more polyclonal and/or monoclonal antibodies, optionallyfreeze-dried or concentrated solutions of an enzyme- orbiotin-conjugated antibody, solutions of an enzyme-avidin conjugate ifbiotin-labeled antibody is used, enzyme substrate in solid or dissolvedform, standard solutions of the melanoma antigen, buffer solutions, and,optionally, polypeptides or detergents for preventing non-specificadsorption and aggregate formation, pipettes, reaction vessels,calibration curves, instruction manuals and the like. One of theantibodies of the test kit is a monoclonal antibody against the melanomaantigen.

G. Monitoring Melanoma Therapy

The antibodies and other affinity molecules of the present invention canalso be used for the determination of a prognostic index that indicatesthe level of melanoma antigen in the body fluids (esp. serum) before,during and following a treatment such as surgery, chemotherapy orradiation therapy. Recurrences of the tumor could be detected by runningtests on fluids for the antigen.

H. Whole Body or Local Imaging

Whole body and local imaging techniques employing radioisotope labelscan be used for locating melanomas, both primary tumors and tumors whichhave metastasized. The antibodies and other affinity molecules of thepresent invention are bound to a suitable radioisotope, typicallytechnetium-99, ¹²³I, ¹²⁵I, or ¹³¹I, or a combination thereof, andadministered to a patient (e.g., parentally). High specific activitylabeling of antibodies or fragments with technetium-99m is described forexample in U.S. Pat. Nos. 5,317,091, 4,478,815, 4,478,818, 4,472,371,Re. 32,417, and 4,311,688. The bio-distribution of the label ismonitored by scintigraphy, and accumulations of the label are related tothe presence of melanoma cells. Whole body imaging techniques aredescribed in U.S. Pat. Nos. 4,036,945 and 4,311,688. Other examples ofagents useful for diagnosis and therapeutic use which can be coupled toantibodies and antibody fragments include metallothionein and fragments(see, U.S. Pat. No. 4,732,864). The disclosures of the cited patents areincorporated herein by reference.

I. Therapeutic Methods

The monoclonal antibodies and other affinity molecules of the presentinvention can be used for combating melanoma cells especially for thepassive immunization of melanoma patients. Thus, the present inventionalso provides a pharmaceutical composition comprising one or moreantibodies or other affinity molecules of the present invention,optionally together with conventional pharmaceutical carrier, adjuvant,filling and additive materials. The pharmaceutical composition can beadministered to melanoma patients.

In a preferred embodiment, the unit dosage of the antibody according tothe present invention for passive immunization is in the range of fromabout 1 to about 200 mg. The unit dosage can be repeatedly administeredto melanoma patients. The monoclonal antibodies can be administeredlocally into the melanoma, see Irie et al., (1986) Proc. Natl. Acad.Sci. USA, 83:8694-8698, or administered systemically into the patient(especially after metastasis). The antibodies according to the presentinvention can be used therapeutically alone or as conjugates withtoxins, radioactive agents, and other therapeutics.

The melanoma antigen can be coated on antigen presenting cells (APC) orbe transfected or pulsed into antigen presenting cells (including, butnot limited to, dentritic cells, B cells, and macrophages) to preparecellular vaccines that can stimulate immune response against melanomacells for treating or preventing melanoma. The isolated melanoma cellscan be fused with antigen presenting cells to prepare cellular vaccinesthat can stimulate immune response against melanoma cells for treatingor preventing melanoma. The above-described cellular vaccines can beaugmented by cytokine treatment and/or attaching a bridge molecule (suchas a bispecific monoclonal antibody) that binds to a costimulatorymolecule (e.g., CD28 or 4-1BB) on T cells using techniques described andcited in PCT publications WO 97/47271, WO 98/16238 and WO 98/04282.

J. Monitoring the Level of Antibodies Against the Melanoma Antigen

The antibodies to the melanoma antigen may themselves be used asantigens for the production of antibodies specific for the idiotypicside of the monoclonal antibody to the melanoma antigen. Theanti-idiotype polyclonal or monoclonal antibody can be used to detectthe presence of antibodies in a host to the melanoma antigen, where theantibody to the melanoma antigen and the physiological fluid to bediagnosed are from the same host. For example, where the antibody to themelanoma antigen is a human antibody or a humanized antibody, thisantibody can itself be used as an antigen to make a monoclonal antibodyspecific for the human idiotype. This anti-idiotype monoclonal antibodycan be used to detect the level of antibodies to the melanoma antigen ina human physiological fluid, for example blood, serum or urine. Theanti-idiotype monoclonal antibody can be made in any host, for examplerodent, more particularly rat or mouse. The anti-idiotype monoclonalantibody can also be a humanized or human antibody.

The anti-idiotype monoclonal antibodies can be used as such or in theform of radioactively labeled derivatives in a radioimmunoassay (RIA).Any of the known modifications of a RIA can be used, for example solublephase (homogeneous) RIA, solid phase (heterogeneous) RIA, single RIA ordouble (sandwich) RIA with direct or indirect (competitive)determination of antibodies directed against melanoma antigen.

In a sandwich RIA, for example, a suitable carrier is coated with ananti-idiotype monoclonal antibody by simple adsorption or optionallyafter activation of the carrier (such as with glutaraldehyde or cyanogenbromide). Suitable carriers include, but are not limited to, the plasticsurface of a microtiter plate or of a test tube (e.g., of polystyrene,polypropylene or polyvinylchloride), glass or plastic beads, filterpaper, dextran, cellulose acetate or nitrocellulose sheets, magneticparticles.

Then test solutions containing antibodies directed against the melanomaantigen and finally radioactively labeled polyclonal antibodies whichalso react with the antibodies directed against the melanoma antigen areadded. The amount of antibodies directed against the melanoma antigen inthe test solution is directly proportional to the amount of boundpolyclonal antibodies and is determined by measuring the radioactivityof the solid phase.

The anti-idiotype monoclonal antibodies can be used as such or in theform of enzyme-conjugated derivatives in an enzyme immunoassay. Any ofthe known modifications of an enzyme immunoassay can be used. The testsare carried out in an analogous manner to the radioimmunoassaysdescribed above using an enzyme label instead of a radioactive label.The amount of immune complex formed which corresponds to the amount ofantibodies directed against the melanoma antigen is determined by addingan enzyme substrate solution. The enzyme substrate reaction results, forexample, in a color change which can be observed by eye or with opticalmeasuring devices.

The anti-idiotype monoclonal antibodies of the present invention can beused as such or in the form of derivatives conjugated withchemiluminescent markers in a chemiluminescence immunoassay. Any of theknown modifications of a chemiluminescence immunoassay can be used. Thetests are carried out in an analogous manner to the radioimmunoassaysdescribed above using a chemiluminescent label instead of a radioactivelabel. The amount of immune complex formed which corresponds to theamount of antibodies directed against the melanoma antigen is determinedby adding a compound triggering luminescence, e.g. H₂O₂ and NaOH, andmeasuring the emission of light with optical measuring devices.

The anti-idiotype monoclonal antibodies and derivatives thereof asdescribed herein before can also be used with other immunoassays,including, but not limited to, immunofluorescence assays, latexagglutination, hemagglutination, evanescent light assays using anoptical fiber coated with an anti-idiotype MAb and other direct-actingimmunosensors which convert the binding event into an electrical oroptical signal, or the like.

The invention also concerns test kits for the qualitative andquantitative determination of antibodies directed against themelanoma-associated antigen comprising anti-idiotype monoclonalantibodies of the invention and/or derivatives thereof and, optionally,other polyclonal or monoclonal antibodies and/or adjuncts.

Test kits according to the invention for a radioimmunoassay contain, forexample, a suitable carrier, optionally freeze-dried solutions of one ormore polyclonal and/or monoclonal antibodies, solutions of aradioactively labeled antibody, standard solutions of antibodiesdirected against the melanoma antigen, buffer solutions, and,optionally, polypeptides or detergents for preventing non-specificadsorption and aggregate formation, pipettes, reaction vessels,calibration curves, instruction manuals and the like. One of theantibodies of the test kit is an anti-idiotype monoclonal antibody.

Test kits according to the invention for an enzyme immunoassay contain,for example, a suitable carrier, optionally freeze-dried solutions ofone or more polyclonal and/or monoclonal antibodies, optionallyfreeze-dried or concentrated solutions of an enzyme- orbiotin-conjugated antibody, solutions of an enzyme-avidin conjugate ifbiotin-labeled antibody is used, enzyme substrate in solid or dissolvedform, standard solutions of antibodies directed against the melanomaantigen, buffer solutions, and, optionally, polypeptides or detergentsfor preventing non-specific adsorption and aggregate formation,pipettes, reaction vessels, calibration curves, instruction manuals andthe like. One of the antibodies of the test kit is an anti-idiotypemonoclonal antibody.

K. Pharmaceutical Formulations and Modes of Administration

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically.Techniques for formulation and administration may be found inRemington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.,Easton, Pa. (1990). Suitable routes may include oral, rectal,transdermal, vaginal, transmucosal, or intestinal administration;parenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections, just to name a few.

Preferred are pharmaceutical compositions for parenteral application.Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Optionally,the suspension may also contain suitable stabilizers or agents whichincrease the solubility of the compounds to allow for the preparation ofhighly concentrated solutions. Exemplary compositions for intramuscular,subcutaneous or intravenous application include isotonic aqueoussolutions or suspensions, optionally prepared shortly before use fromlyophilized or concentrated preparations. The pharmaceuticalcompositions may contain agents for conserving, stabilizing, wetting,emulsifying or solubilizing the ingredients, salts for the regulation ofthe osmotic pressure, buffer and/or compounds regulating the viscosity(e.g., sodium carboxycellulose, sorbitol, dextran, polyvinylpyrrolidineor gelatine). They are prepared by methods known in the art, e.g. byconventional mixing, dissolving or lyophilizing, and contain from about0.01% to about 50% of active ingredients. The compositions forinjections are processed, filled into ampoules or vials, and sealedunder aseptic conditions according to methods known in the art.

The specific mode of administration and the dosage will be selected bythe attending physician taking into account the particulars of thepatient, the state of the disease, the type of melanoma treated, and thelike. It should be noted that the attending physician would know how toand when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministrated dose in the management of the disorder of interest willvary with the severity of the condition to be treated and to the routeof administration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

By “comprising” is meant including, but not limited to, whatever followsthe word “comprising”. Thus, use of the term “comprising” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and may or may not be present. By “consisting of” is meantincluding, and limited to, whatever follows the phrase “consisting of”.Thus, the phrase “consisting of” indicates that the listed elements arerequired or mandatory, and that no other elements may be present. By“consisting essentially of” is meant including any elements listed afterthe phrase, and limited to other elements that do not interfere with orcontribute to the activity or action specified in the disclosure for thelisted elements. Thus, the phrase “consisting essentially of” indicatesthat the listed elements are required or mandatory, but that otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

Other features and advantages of the invention will be apparent from thedetailed description of the invention below and from the list ofenumerated embodiments that follows.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1D show immunohistochemical-staining of melanocytic lesionswith monoclonal antibody SM5-1.

FIG. 1A shows homogenous staining of compound nevus cells. Thenon-activated basal melanocytes are not stained.

FIG. 1B shows in-situ staining of melanoma.

FIG. 1C shows staining of superficial spreading melanoma (Clark levelIII, Breslow 0.91 mm).

FIG. 1D shows staining of melanoma lymph node metastasis.

SM5-1 stained the entire population of tumor cells in 1B-1D.

V. DETAILED DESCRIPTION OF THE INVENTION

A. Preparation of SM5-1 Antibody

SM5-1 antibody is selected from a panel of monoclonal antibodiesgenerated by using a subtractive imnunization protocol as previouslydescribed in Williams et al., (1992) Biotechniques 12:842-8477, andBrooks et al., (1993) J. Cell Biol. 22:1351-1359, both of which areincorporated by reference herein.

Briefly, mice were immunized with human melanoma cell line SMMU-1, whichwas obtained from the primary melanoma of a patient (Guo et al., (1994)Cancer Res. 54:561-1565, incorporated by reference herein). Next, themice were treated with cyclophosphamide to abrogate activated B cellsthat produce antibody against epitopes expressed by the primarymelanoma. The mice were then immunized with human melanoma cell lineSMMU-2, which was obtained from the metastatic lesion of the samepatient. The splenocytes from the mice were used for making hybridomasusing standard techniques. Köhler G, Milstein C: Derivation of specificantibody producing tissue culture and tumor lines by cell fusion. Eur.J. Immunol. 1976; 6:511-519.

B. Immuno-Histochemical Staining of Tissue Specimen

SM5-1 was determined by ELISA to be a mouse IgG1. SM5-1 wascharacterized for its reactivity with both frozen and paraffin embeddedtissue samples. Supernatants from SM5-1 hybridoma were harvested andused for immuno-histochemical staining of tissues using the labeledstreptavidin-biotin (LSAB) method (Large Volume LSAB kit, DAKO,Carpinteria, Calif., USA) as described elsewhere. Giomo R A: Acomparison of two immunoperoxidase staining methods based on theavidin-biotin interaction. Diag. Immunol. 1984; 2:161-166.

Briefly, the specimens were incubated with the primary antibody followedby 10-minute incubations with biotinylated link antibody,peroxidase-labeled streptavidin and substrate chromogen-solution. Forall experiments 1:50 diluted tissue culture supernatants of SM5-1 wereused. Staining for SM5-1 was improved with pretreatment of tissuesections in a microwave oven and with trypsin. Shi S R, Key M E, Kalra KL: Antigen retrieval in formalin-fixed, paraffin-embedded tissues: anenhancement method for immuno-histochemical staining based on microwaveoven heating of tissue sections. J. Histochem. Cytochem. 1991;39:741-748.

Samples of melanocytic lesions were also stained with HMB-45 (1:50,DAKO) and with a monoclonal antibody to S-100 protein (clone S 1/61/699,1:20, Medac).

226 consecutive specimens of human melanoma were selected for staining,including 88 primary melanomas and 138 metastases (skin, lymph nodes).16 nevi of various histological classifications (see Table 1) wereselected for staining as non-melanoma melanocytic lesions specimens.TABLE 1 Reactivity of SM5-1 with benign and malignant melanocyticlesions in comparison with HMB-45 and anti-S-100 on paraffin embeddedsections Immunohistochemical staining with Melanocytic SM5-1 HMB-45Anti-S-100 lesions (positive/tested) (positive/tested) (positive/tested)NEVI Compound  6/6  6/6  6/6 Dysplastic  5/5  5/5  5/5 Blue  2/2  2/2 2/2 Combined  3/3  3/3  3/3 Total  16/16 (100%)  16/16 (100%)  16/16(100%) MELANOMA Primary melanoma MM in situ  9/9  9/9  9/9 Clark levelII  7/7  7/7  7/7 Clark level III  19/19  19/19  19/19 Clark level IV 47/47  46/47  47/47 Clark level V  6/6  6/6  6/6 Total  88/88 (100%) 87/88 (98%)  88/88 (100%) Metastatic 135/138 (100%) 114/138 (83%)137/138 (100%) melanoma Total for 223/226 (98%) 201/226 (88%) 225/226(99%) Melanoma

The tissues were surgically removed, fixed in 10% buffered formaldehyde,embedded in paraffin, and processed for routine histology study.

Specimens of non-melanocytic tumors and benign tissues are listed inTable 2 and Table 3, some of which were provided by the Institute ofPathology, Charité, Germany. TABLE 2 Reactivity of SM5-1 withnon-melanocytic tumors on paraffin embedded sections.Immunohistochemical Tumor type staining with SM5-1 SkinDermatofibrosarcoma protuberans 0/2 Merkel cell carcinoma 0/1 Sweatgland carcinoma 0/3 Sqnamous cell carcinoma 0/4 Brain Glioma 0/1Astrocytoma 0/4 Oligodendroglioma 0/1 Meningeoma 0/3 Glioblastoma 0/2Neurinoma 0/1 Gastrointestinal tract Esophageal carcinoma 0/2 Gastriccarcinoma 0/2 Colon carcinoma 0/2 Pancreatic carcinoma 0/2 OthersBronchial carcinoma 0/1 Thyroid carcinoma 0/1 Breast carcinoma 0/1Prostate carcinoma 0/1 Ovary carcinoma 0/1 Renal cell carcinoma 0/1Leiomyosarcoma 0/2 M. Paget 0/1 Angioleiomyosarcoma 0/1 Angiosarcoma 0/3Porocarcinoma 0/1 Urothel carcinoma 0/1 Osteosarcoma 0/2 Total  0/46

C. SM5-1 Recognizes an Antigen in Malignant Melanoma and Nevi

Table 1 summarizes immuno-histochemical analyses of 226 melanomas and 16nevi with SM5-1, HMB-45 and anti-S 100.

All of the tested primary melanoma and nevi were positive for SM5-1irrespective of their histological type (superficial spreading, nodular,lentigo maligna, acral lentiginous) or Clark level. Generally, thestaining was strong and involved the majority of the neoplastic cells.The pattern of the staining was both cytoplasmic and on the cellmembrane. There was no difference in the strength or pattern of stainingbetween primary lesions and metastatic lesions (FIG. 1).

HMB-45 was able to detect all of the examined primary melanoma and nevi.However, HMB-45 was only able to stain 83% (114/138) of metastaticmelanoma. Most interestingly, all the HMB-45 negative melanomas werestained by SM5-1. In tissue samples stained by both SM5-1 and HMB-45,the epitopes for SM5-1 and for HMB-45 were found in differentpopulations of the tumor. This indicates that the epitopes for SM5-1 andHMB-45 are different and, furthermore, they are expressed in differentpopulations of melanoma tissue samples.

D. SM5-1 Reacted with Frozen Sections of Melanoma

36 melanoma specimens (17 primary melanomas, 19 metastases) obtainedfrom surgical resection were split in half with one part being paraffinembedded and the other part snap frozen (snap frozen in liquid nitrogenand cut with a freezing microtome (5 μm)) and stained with SM5-1(dilution 1:50) on cryostat sections. The cryostat sections wereair-dried and fixed in acetone. All frozen sections reacted with SM5-1in a pattern comparable with that seen in paraffin section.

E. SM5-1 Recognizes an Antigen in Human Hepatocellular Carcinoma CellLines

7 HCC cell lines established from human hepatocellular carcinoma tissueresected between August 1994 to December 1996 were tested by SM5-1. Allof the HCC cells grow stably in RPMI-1640 medium supplemented with 10%FCS. Human B cells transfected with EBV were used as a negative control.

Hepatocellular cell lines were trypsinized to form single cellsuspensions and washed with PBS, then 0.1 ml SM5-1 supernatant was addedto 10⁵ tumor cells, incubated at 4° C. for 1 h, washed with PBS twice,then 0.1 ml FITC-GaM-IgG (FITC labeled Goat anti-mouse IgG (ZYMED, SanFrancisco), 1:50 in PBS) was added. After 40 minutes of incubation at 4°C., cells were washed with PBS twice. The samples were assayed at anFACSCaliber® cytometer. 6/7 of the hepatoma cell lines were stainedpositive by SM5-1.

An antigen was immuno-precipitated by SM5-1 antibody from the extractsof the HCC with the strongest staining signal as a protein of about 200kilodalton (kD) apparent molecular weight as determined by sodiumdodecyl sulfate gel electrophoresis.

F. SM5-1 does not Stain Non-melanocytic Malignant Tumors

In order, to examine whether SM5-1 cross-reacts with malignancies otherthan melanomas, a large variety of paraffin embedded non-melanoma tumors(surgically removed) were examined for staining with SM5-1. Thesetissues included tumors from skin, brain, gastrointestinal tract andothers. Table 2 shows that none of the 27 non-melanocytic tumors reactedwith SM5-1.

G. SM5-1 does not Stain Most Normal Human Tissues

Table 3 summarizes staining data with nonmalignant human tissues. Mostnormal cells were not stained with SM5-1 except for weak staining withplasma cells, the secretion of eccrine sweat gland, the secretoryepithelium of thyroid, some myofibroblasts, and perivascular dendriticcells. This limited pattern of unspecific staining is comparable to theunspecific staining by HMB-45. Colombari R, Bonetti F, Zamboni G, ScarpaA, Marino F, Tomezzoli A et al: Distribution of melanoma specificantibody (HMB-45) in benign and malignant melanocytic tumors. VirchowsArchiv. A Pathol. Anat. 1988; 413:17-24. TABLE 3 Reactivity of SM5-1with normal tissues, normal cells and benign lesions immunohistochemicalstaining Tissue or lesion with SM5-1 NEGATIVE Glomerulus −−− Ciliatedepithelium −−− Beaker cells −−− Sebacious glands −−− Liver tissue −−−Normal melanocytes −−− Keratinocytes −−− Langerhans cells −−− Peripheralnerves −−− Neutrophilic granulocytes −−− Smooth muscle cells −−− Gastricmucosa −−− Ependymoma −−− Keloid −−− Activated melanocytes −−− WEAKLYPOSITIVE Activated macrophages + Myofibroblasts + Plasma cells +Secretion of eccrine sweat glands + Secretory epithelium of thyroid +

H. Staining Pattern of SM5-1

With regard to the staining pattern we found that HMB-45 staining iscytoplasmic and granular whereas for SM5-1 it is more homogenous andboth cytoplasmic and on the cell membrane. This suggests that the twoantibodies recognize different antigens.

The results show that SM5-1 is more sensitive than HMB-45 and morespecific than anti-S-100 antibodies in detecting melanoma lesions.

I. Utility of SM5-1

Melanoma patients have a significantly increased risk of developing asecond primary tumor, e.g. lung cancer. Swerdlow A J, Storm H H, SasieniP D: Risks of second primary malignancy in patients with cutaneous andocular melanoma in Denmark, 1943-1989. Int. J. Cancer 1995; 61:773-779.Therefore, it is of great importance to analyze and identify any lesionor suspected parenchymal lesion in melanoma patients, e.g., by fineneedle aspiration. The ability of SM5-1 to reliably recognize metastasesof malignant melanoma in patients bearing poorly differentiated tumorsis very useful in such procedures. In one series of cases a secondpulmonary neoplasia could be identified in three of 50 melanoma patientsby using two different antibodies for the immunocytochemical diagnosis.Mottolese M, Venturo I, Benevolo M, Di-Filippo F, Lopez M, Bigotti A etal: Immunocytochemical diagnosis of amelanotic metastatic melanoma usingmonoclonal antibodies HMB-45 and Ep 1-3. Melanoma Res. 1994; 4:53-58.Using SM5-1 can render it unnecessary to use a panel of antibodies insuch cases.

By using SM5-1 one can correctly diagnose a lesion as being ofmelanocytic origin. The differentiation between a nevus and a melanomacan usually then be done with relative ease by histological criteria.SM5-1 can be used in routinely processed paraffin embedded or cryostatmelanoma sections.

All publications cited in the specification are incorporated byreference herein, including drawings and sequences listed in eachpublication. All the compounds disclosed and referred to in thepublications mentioned above are incorporated by reference herein,including those compounds disclosed and referred to in articles cited bythe publications mentioned above.

Other embodiments of this invention are disclosed in the followingclaims.

1. A monoclonal antibody which specifically binds to an antigen on humanmelanoma cells, wherein (a) said antigen is specifically bound by theantibody produced by the hybridoma deposited under ATCC Accession No.12588, (b) said antigen is present on the membrane and in the cytoplasmof human melanoma cells, and (c) said antigen is not present in normalnon-activated human melanocytic cells and non-melanocytic human tumorcells in an amount that is detectable by the antibody produced by thehybridoma deposited under ATCC Accession No.
 12588. 2-18. (canceled)